Neuropeptide Y (NPY) and catestatin peptides are secreted from adrenomedullary chromaffin cells and sympathetic nerves for the regulation of blood pressure. NPY acts as a direct vasoconstrictor, and catestatin functions as an autocrine regulator to inhibit nicotine-stimulated catecholamine release. Elevated NPY and reduced catestatin in essential hypertension implicate their participation as neuroeffectors in regulating blood pressure. Importantly, knowledge of the major proteolytic enzymes (s) responsible for converting their respective prohormone precursors into active NPY and catestatin is crucial for understanding regulatory mechanisms that control blood pressure. The prohormone precursors of NPY and catestatin, pro-NPY and chromogranin A (CgA), respectively, undergo proteolytic processing within secretory vesicles of adrenal medulla, known as chromaffin granules. We have identified secretory vesicle cathepsin L, previously known as ?prohormone thiol protease? (PTP), as a key processing enzyme for pro-NPY and CgA. Moreover, this project discovered a novel endogenous serpin, endopin 2 that inhibits secretory vesicle cathepsin L. In addition, the subtilisin-like PC1 and PC2 proteases in secretory vesicles may also participate in pro-NPY and CgA processing. Based on these new findings, the goal of Project 3 will be to assess the roles of secretory vesicle cathepsin L and endopin 2, compared to PC1 and PC2, in the production of NPY and catestatin neuropeptides that regulate blood pressure. This project will test the hypothesis that secretory vesicle cathepsin L may be a major processing enzyme for NPY and catestatin, compared to PC1 and PC2 enzymes. Our new results support the emerging biological role of cathepsin L function in secretory vesicles for proteolysis of pro-NPY and CgA. Moreover, our recent studies of cathepsin L knockout mice suggest participation of this protease in NPY production in adrenals. These results lead to the next phase of this study that will (1) evaluate in vitro and cellular processing of pro-NPY and CgA by secretory vesicle cathepsin L, compared to PC1 and PC2, (2) assess the cellular and tissue distribution of cathepsin L and PC enzymes in secretory vesicles that contain NPY and catestatin, (3) conduct cellular antisense and gene knockout studies to examine the relative roles of cathepsin L and PC enzymes for NPY and catestatin production, and (4) evaluate endopin 2 as an endogenous serpin inhibitor of cathepsin L for neuropeptide production. Results will demonstrate the relative roles for cathepsin L and endopin 2, compared to PC1 and PC2, in the biosynthesis of active NPY and catestatin peptide regulators. Project 3 complements the program project theme of understanding the regulation of sympathetic neuroeffectors that participate in blood pressure regulation.